Actin regulatory elements for use in plants

ABSTRACT

The present invention provides polynucleotide molecules isolated from  Oryza sativa  and  Zea mays  and useful for expressing transgenes in plants. The present invention also provides expression constructs containing the polynucleotide molecules useful for expressing transgenes in plants. The present invention also provides transgenic plants and seeds containing the polynucleotide molecules useful for expressing transgenes in plants.

This application is a continuation of U.S. application Ser. No.11/801,137, filed May 8, 2007; now U.S. Pat. No. 7,807,812 whichapplication is a Divisional of U.S. patent application Ser. No.10/950,233, filed 24 Sep. 2004 now U.S. Pat. No. 7,408,054 and publishedas U.S. Patent Application Publication US20060162010A1, which claimspriority to U.S. Provisional Patent Application No. 60/505,949, filed 25Sep., 2003, each of the disclosures of which are incorporated byreference in their entirety herein.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Seq. Listing Copy 1 and Seq. ListingCopy 2) and a computer-readable form of the sequence listing, all onCD-ROMs, each containing the file named pa_(—)01298.rpt, which is 16,783bytes (measured in MS Windows®) and was created on May 5, 2007, arehereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to the field of plant molecular biology and plantgenetic engineering and polynucleotide molecules useful for theexpression of transgenes in plants.

BACKGROUND

One of the goals of plant genetic engineering is to produce plants withagronomically desirable characteristics or traits. The proper expressionof a desirable transgene in a transgenic plant is one way to achievethis goal. Regulatory elements such as promoters, leaders, and intronsare non-coding polynucleotide molecules which play an integral part inthe overall expression of genes in living cells. Isolated regulatoryelements that function in plants are therefore useful for modifyingplant phenotypes through the methods of genetic engineering.

Many regulatory elements are available and are useful for providing goodoverall expression of a transgene. For example, constitutive promoterssuch as P-FMV, the promoter from the 35S transcript of the Figwortmosaic virus (U.S. Pat. No. 6,051,753); P-CaMV 35S, the promoter fromthe 35S RNA transcript of the Cauliflower mosaic virus (U.S. Pat. No.5,530,196); P-Rice Actin 1, the promoter from the actin 1 gene of Oryzasativa (U.S. Pat. No. 5,641,876); and P-NOS, the promoter from thenopaline synthase gene of Agrobacterium tumefaciens are known to providesome level of gene expression in most or all of the tissues of a plantduring most or all of the plant's lifespan. While previous work hasprovided a number of regulatory elements useful to affect geneexpression in transgenic plants, there is still a great need for novelregulatory elements with beneficial expression characteristics. Manypreviously identified regulatory elements fail to provide the patternsor levels of expression required to fully realize the benefits ofexpression of selected genes in transgenic crop plants.

Spatial organization within the eukaryotic cell and directed movementsof the cell contents are mediated by the cytoskeleton, a network offilamentous protein polymers that permeates the cytosol. Actin is one ofthe three major families of proteins making up the cytoskeleton. Membersof this multi-gene family have been reported in almost all eukaryoticspecies including yeast, humans, mouse, Drosophila, tobacco, maize,rice, soybean, potato and Arabidopsis. Plant actins are encoded by amulti-gene family, constituted by a number of different isotypes.

We hypothesized that the regulatory elements from an actin gene mighthave a constitutive expression pattern and that the regulatory elementscould be useful to direct expression of a transgene such as a glyphosateresistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) transgeneto produce a glyphosate tolerant plant. The efficient production ofglyphosate tolerant plants requires the use of regulatory elementscapable of directing transgene expression in all tissues including themost sensitive reproductive organs such as anthers and meristem tissues.The present invention thus provides such regulatory elements isolatedfrom actin genes of Oryza sativa and Zea mays.

SUMMARY

In one embodiment the invention provides polynucleotide moleculesisolated from Oryza sativa and Zea mays useful for modulating transgeneexpression in plants. In another embodiment the invention providesexpression constructs containing the polynucleotide molecules useful formodulating transgene expression in plants. In another embodiment theinvention provides transgenic plants and seeds containing thepolynucleotide molecules useful for modulating transgene expression inplants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Construct pMON54990 containing the P-Os-Act15a cassette.

FIG. 2: Construct pMON54976 containing the P-Os-Act15b cassette.

FIG. 3: Construct pMON54991 containing the P-Os-Act16 cassette.

FIG. 4: Construct pMON54977 containing the P-Os-Act18 cassette.

FIG. 5: Construct pMON54978 containing the P-Os-Act31 cassette.

FIG. 6: Construct pMON54980 containing the P-Zm-Act31 cassette.

FIG. 7: Construct pMON54981 containing the P-Zm-Act33 cassette.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

The invention disclosed herein provides polynucleotide molecules havinggene regulatory activity from Oryza sativa and Zea mays. The design,construction, and use of these polynucleotide molecules are one objectof this invention. The polynucleotide sequences of these polynucleotidemolecules are provided as SEQ ID NO: 1-7. These polynucleotide moleculesare capable of affecting the transcription of operably linkedtranscribable polynucleotide molecules in both vegetative andreproductive tissues of plants and therefore can selectively regulateexpression of transgenes in these tissues.

As used herein, the term “polynucleotide molecule” refers to the single-or double-stranded DNA or RNA of genomic or synthetic origin, i.e., apolymer of deoxyribonucleotide or ribonucleotide bases, respectively,read from the 5′ (upstream) end to the 3′ (downstream) end.

As used herein, the term “polynucleotide sequence” refers to thesequence of a polynucleotide molecule. The nomenclature for DNA bases asset forth at 37 CFR §1.822 is used.

As used herein, the term “gene regulatory activity” refers to apolynucleotide molecule capable of affecting transcription ortranslation of an operably linked transcribable polynucleotide molecule.An isolated polynucleotide molecule having gene regulatory activity mayprovide temporal or spatial expression or modulate levels and rates ofexpression of the operably linked transcribable polynucleotide molecule.An isolated polynucleotide molecule having gene regulatory activity maycomprise a promoter, intron, leader, or 3′ transcriptional terminationregion.

As used herein, the term “regulatory element” refers to an isolatedpolynucleotide molecule capable of having gene regulatory activity. Aregulatory element may comprise a promoter, intron, leader, or 3′transcriptional termination region.

As used herein, the term “promoter” refers to a polynucleotide moleculethat is involved in recognition and binding of RNA polymerase II andother proteins (trans-acting transcription factors) to initiatetranscription. A “plant promoter” is a native or non-native promoterthat is functional in plant cells. A plant promoter can be used as a 5′regulatory element for modulating expression of an operably linked geneor genes. Plant promoters may be defined by their temporal, spatial, ordevelopmental expression pattern.

A promoter comprises subfragments that have promoter activity.Subfragments may comprise enhancer domains and may be useful forconstructing chimeric promoters. Subfragments of SEQ ID NO: 1 compriseat least about 75, 85, 90, 95, 110, 125, 250, 400, 750, 1000, 1300,1500, and 1700 contiguous nucleotides of the polynucleotide sequence ofSEQ ID NO: 1-7. Subfragments of SEQ ID NO: 2 comprise at least about 95,110, 125, 250, 400, 750, 1000, and 1300 contiguous nucleotides of thepolynucleotide sequence of SEQ ID NO: 2. Subfragments of SEQ ID NO: 3comprise at least about 95, 110, 125, 250, 400, 750, 1000, 1300, 1500,and 1800 contiguous nucleotides of the polynucleotide sequence of SEQ IDNO: 3. Subfragments of SEQ ID NO: 4 comprise at least about 95, 110,125, 250, 400, 750, and 1000 contiguous nucleotides of thepolynucleotide sequence of SEQ ID NO: 4. Subfragments of SEQ ID NO: 5comprise at least about 95, 110, 125, 250, 400, 750, 1000, 1300, 1500,1800, and 2500 contiguous nucleotides of the polynucleotide sequence ofSEQ ID NO: 5. Subfragments of SEQ ID NO: 6 comprise at least about 95,110, 125, 250, 400, 750, 1000, 1300, and 1500 contiguous nucleotides ofthe polynucleotide sequence of SEQ ID NO: 6. Subfragments of SEQ ID NO:7 comprise at least about 95, 110, 125, 250, 400, 750, 1000, and 1300contiguous nucleotides of the polynucleotide sequence of SEQ ID NO: 7.

As used herein, the term “enhancer domain” refers to a cis-actingtranscriptional regulatory element, a.k.a. cis-element, which confers anaspect of the overall control of gene expression. An enhancer domain mayfunction to bind transcription factors, trans-acting protein factorsthat regulate transcription. Some enhancer domains bind more than onetranscription factor, and transcription factors may interact withdifferent affinities with more than one enhancer domain. Enhancerdomains can be identified by a number of techniques, including deletionanalysis, i.e., deleting one or more nucleotides from the 5′ end orinternal to a promoter; DNA binding protein analysis using DNase Ifootprinting, methylation interference, electrophoresis mobility-shiftassays, in vivo genomic footprinting by ligation-mediated PCR, and otherconventional assays; or by DNA sequence similarity analysis with knowncis-element motifs by conventional DNA sequence comparison methods. Thefine structure of an enhancer domain can be further studied bymutagenesis (or substitution) of one or more nucleotides or by otherconventional methods. Enhancer domains can be obtained by chemicalsynthesis or by isolation from promoters that include such elements, andthey can be synthesized with additional flanking nucleotides thatcontain useful restriction enzyme sites to facilitate subsequencemanipulation. Thus, the design, construction, and use of enhancerdomains according to the methods disclosed herein for modulating theexpression of operably linked polynucleotide molecules are encompassedby the present invention.

As used herein, the term “chimeric” refers to the product of the fusionof portions of two or more different polynucleotide molecules. As usedherein, the term “chimeric promoter” refers to a promoter producedthrough the manipulation of known promoters or other polynucleotidemolecules. Such chimeric promoters may combine enhancer domains that canconfer or modulate gene expression from one or more promoters, forexample, by fusing a heterologous enhancer domain from a first promoterto a second promoter with its own partial or complete regulatoryelements. Thus, the design, construction, and use of chimeric promotersaccording to the methods disclosed herein for modulating the expressionof operably linked polynucleotide molecules are encompassed by thepresent invention.

As used herein, the term “percent sequence identity” refers to thepercentage of identical nucleotides in a linear polynucleotide sequenceof a reference polynucleotide molecule (or its complementary strand) ascompared to a test polynucleotide molecule (or its complementary strand)when the two sequences are optimally aligned (with appropriatenucleotide insertions, deletions, or gaps totaling less than 20 percentof the reference sequence over the window of comparison). Optimalalignment of sequences for aligning a comparison window are well knownto those skilled in the art and may be conducted by tools such as thelocal homology algorithm of Smith and Waterman, the homology alignmentalgorithm of Needleman and Wunsch, the search for similarity method ofPearson and Lipman, and preferably by computerized implementations ofthese algorithms such as GAP, BESTFIT, FASTA, and TFASTA available aspart of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, Mass.).An “identity fraction” for aligned segments of a test sequence and areference sequence is the number of identical components which areshared by the two aligned sequences divided by the total number ofcomponents in the reference sequence segment, i.e., the entire referencesequence or a smaller defined part of the reference sequence. Percentsequence identity is represented as the identity fraction multiplied by100. The comparison of one or more polynucleotide sequences may be to afull-length polynucleotide sequence or a portion thereof, or to a longerpolynucleotide sequence.

As used herein, the term “substantial percent sequence identity” refersto a percent sequence identity of at least about 70% sequence identity,at least about 80% sequence identity, at least about 90% sequenceidentity, or even greater sequence identity, such as about 98% or about99% sequence identity. Thus, one embodiment of the invention is apolynucleotide molecule that has at least about 70% sequence identity,at least about 80% sequence identity, at least about 90% sequenceidentity, or even greater sequence identity, such as about 98% or about99% sequence identity with a polynucleotide sequence described herein.Polynucleotide molecules that are capable of regulating transcription ofoperably linked transcribable polynucleotide molecules and have asubstantial percent sequence identity to the polynucleotide sequences ofthe polynucleotide molecules provided herein are encompassed within thescope of this invention.

Promoter Isolation and Modification Methods

Any number of methods well known to those skilled in the art can be usedto isolate fragments of a promoter disclosed herein. For example, PCR(polymerase chain reaction) technology can be used to amplify flankingregions from a genomic library of a plant using publicly availablesequence information. A number of methods are known to those of skill inthe art to amplify unknown polynucleotide molecules adjacent to a coreregion of known polynucleotide sequence. Methods include but are notlimited to inverse PCR (IPCR), vectorette PCR, Y-shaped PCR, and genomewalking approaches. Polynucleotide fragments can also be obtained byother techniques such as by directly synthesizing the fragment bychemical means, as is commonly practiced by using an automatedoligonucleotide synthesizer. For the present invention, thepolynucleotide molecules were isolated from genomic DNA by designing PCRprimers based on available sequence information.

Novel chimeric promoters can be designed or engineered by a number ofmethods. For example, a chimeric promoter may be produced by fusing anenhancer domain from a first promoter to a second promoter. Theresultant chimeric promoter may have novel expression propertiesrelative to the first or second promoters. Novel chimeric promoters canbe constructed such that the enhancer domain from a first promoter isfused at the 5′ end, at the 3′ end, or at any position internal to thesecond promoter. The location of the enhancer domain fusion relative tothe second promoter may cause the resultant chimeric promoter to havenovel expression properties relative to a fusion made at a differentlocation.

Those of skill in the art are familiar with the standard resourcematerials that describe specific conditions and procedures for theconstruction, manipulation, and isolation of macromolecules (e.g.,polynucleotide molecules, plasmids, etc.), as well as the generation ofrecombinant organisms and the screening and isolation of polynucleotidemolecules.

Constructs

As used herein, the term “construct” refers to any recombinantpolynucleotide molecule such as a plasmid, cosmid, virus, autonomouslyreplicating polynucleotide molecule, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA polynucleotide molecule,derived from any source, capable of genomic integration or autonomousreplication, comprising a polynucleotide molecule where one or morepolynucleotide molecule has been operably linked.

As used herein, the term “operably linked” refers to a firstpolynucleotide molecule, such as a promoter, connected with a secondtranscribable polynucleotide molecule, such as a gene of interest, wherethe polynucleotide molecules are so arranged that the firstpolynucleotide molecule affects the function of the secondpolynucleotide molecule. The two polynucleotide molecules may be part ofa single contiguous polynucleotide molecule and may be adjacent. Forexample, a promoter is operably linked to a gene of interest if thepromoter regulates or mediates transcription of the gene of interest ina cell.

As used herein, the term “transcribable polynucleotide molecule” refersto any polynucleotide molecule capable of being transcribed into a RNAmolecule. Methods are known for introducing constructs into a cell insuch a manner that the transcribable polynucleotide molecule istranscribed into a functional mRNA molecule that is translated andtherefore expressed as a protein product. Constructs may also beconstructed to be capable of expressing antisense RNA molecules, inorder to inhibit translation of a specific RNA molecule of interest. Forthe practice of the present invention, conventional compositions andmethods for preparing and using constructs and host cells are well knownto one skilled in the art, see for example, Molecular Cloning: ALaboratory Manual, 3^(rd) edition Volumes 1, 2, and 3 (2000) J. F.Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor LaboratoryPress.

Constructs of the present invention would typically contain a promoteroperably linked to a transcribable polynucleotide molecule operablylinked to a 3′ transcription termination polynucleotide molecule. Inaddition, constructs may include but are not limited to additionalregulatory polynucleotide molecules from the 3′-untranslated region (3′UTR) of plant genes (e.g., a 3′ UTR to increase mRNA stability of themRNA, such as the PI-II termination region of potato or the octopine ornopaline synthase 3′ termination regions). Constructs may include butare not limited to the 5′ untranslated regions (5′ UTR) of an mRNApolynucleotide molecule which can play an important role in translationinitiation and can also be a genetic component in a plant expressionconstruct. For example, non-translated 5′ leader polynucleotidemolecules derived from heat shock protein genes have been demonstratedto enhance gene expression in plants (see for example, U.S. Pat. No.5,659,122 and U.S. Pat. No. 5,362,865, all of which are herebyincorporated by reference). These additional upstream and downstreamregulatory polynucleotide molecules may be derived from a source that isnative or heterologous with respect to the other elements present on thepromoter construct.

Thus, one embodiment of the invention is a promoter such as provided inSEQ ID NO: 1-7, operably linked to a transcribable polynucleotidemolecule so as to direct transcription of said transcribablepolynucleotide molecule at a desired level or in a desired tissue ordevelopmental pattern upon introduction of said construct into a plantcell. In some cases, the transcribable polynucleotide molecule comprisesa protein-coding region of a gene, and the promoter provides fortranscription of a functional mRNA molecule that is translated andexpressed as a protein product. Constructs may also be constructed fortranscription of antisense RNA molecules or other similar inhibitory RNAin order to inhibit expression of a specific RNA molecule of interest ina target host cell.

Exemplary transcribable polynucleotide molecules for incorporation intoconstructs of the present invention include, for example, polynucleotidemolecules or genes from a species other than the target gene species, oreven genes that originate with or are present in the same species, butare incorporated into recipient cells by genetic engineering methodsrather than classical reproduction or breeding techniques. Exogenousgene or genetic element is intended to refer to any gene orpolynucleotide molecule that is introduced into a recipient cell. Thetype of polynucleotide molecule included in the exogenous polynucleotidemolecule can include a polynucleotide molecule that is already presentin the plant cell, a polynucleotide molecule from another plant, apolynucleotide molecule from a different organism, or a polynucleotidemolecule generated externally, such as a polynucleotide moleculecontaining an antisense message of a gene, or a polynucleotide moleculeencoding an artificial or modified version of a gene.

The promoters of the present invention can be incorporated into aconstruct using marker genes as described and tested in transientanalyses that provide an indication of gene expression in stable plantsystems. As used herein the term “marker gene” refers to anytranscribable polynucleotide molecule whose expression can be screenedfor or scored in some way. Methods of testing for marker gene expressionin transient assays are known to those of skill in the art. Transientexpression of marker genes has been reported using a variety of plants,tissues, and DNA delivery systems. For example, types of transientanalyses can include but are not limited to direct gene delivery viaelectroporation or particle bombardment of tissues in any transientplant assay using any plant species of interest. Such transient systemswould include but are not limited to electroporation of protoplasts froma variety of tissue sources or particle bombardment of specific tissuesof interest. The present invention encompasses the use of any transientexpression system to evaluate promoters or promoter fragments operablylinked to any transcribable polynucleotide molecules, including but notlimited to selected reporter genes, marker genes, or genes of agronomicinterest. Examples of plant tissues envisioned to test in transients viaan appropriate delivery system would include but are not limited to leafbase tissues, callus, cotyledons, roots, endosperm, embryos, floraltissue, pollen, and epidermal tissue.

Any scorable or screenable marker gene can be used in a transient assay.Exemplary marker genes for transient analyses of the promoters orpromoter fragments of the present invention include a GUS gene (U.S.Pat. No. 5,599,670, hereby incorporated by reference) or a GFP gene(U.S. Pat. No. 5,491,084 and U.S. Pat. No. 6,146,826, both of which arehereby incorporated by reference). The constructs containing thepromoters or promoter fragments operably linked to a marker gene aredelivered to the tissues and the tissues are analyzed by the appropriatemechanism, depending on the marker. The quantitative or qualitativeanalyses are used as a tool to evaluate the potential expression profileof the promoters or promoter fragments when operatively linked to genesof agronomic interest in stable plants.

Thus, in one preferred embodiment, a polynucleotide molecule of thepresent invention as shown in SEQ ID NO: 1-7 is incorporated into a DNAconstruct such that a polynucleotide molecule of the present inventionis operably linked to a transcribable polynucleotide molecule thatprovides for a selectable, screenable, or scorable marker. Markers foruse in the practice of the present invention include, but are notlimited to transcribable polynucleotide molecules encodingβ-glucuronidase (GUS), green fluorescent protein (GFP), luciferase(LUC), proteins that confer antibiotic resistance, or proteins thatconfer herbicide tolerance. Useful antibiotic resistance markers,including those encoding proteins conferring resistance to kanamycin(nptII), hygromycin B (aph IV), streptomycin or spectinomycin (aad,spec/strep) and gentamycin (aac3 and aacC4) are known in the art.Herbicides for which transgenic plant tolerance has been demonstratedand the method of the present invention can be applied, include but arenot limited to: glyphosate, glufosinate, sulfonylureas, imidazolinones,bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidaseinhibitors, and isoxasflutole herbicides. Polynucleotide moleculesencoding proteins involved in herbicide tolerance are known in the art,and include, but are not limited to a polynucleotide molecule encoding5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) described in U.S.Pat. No. 5,627,061, U.S. Pat. No. 5,633,435, U.S. Pat. No. 6,040,497 andin U.S. Pat. No. 5,094,945 for glyphosate tolerance, all of which arehereby incorporated by reference; polynucleotides encoding a glyphosateoxidoreductase and a glyphosate-N-acetyl transferase (GOX, U.S. Pat. No.5,463,175 and GAT, U.S. Patent publication 20030083480, hereinincorporated by reference); a polynucleotide molecule encodingbromoxynil nitrilase (Bxn) described in U.S. Pat. No. 4,810,648 forBromoxynil tolerance, which is hereby incorporated by reference; apolynucleotide molecule encoding phytoene desaturase (crtI) described inMisawa et al, (1993) Plant J. 4:833-840 and Misawa et al, (1994) PlantJ. 6:481-489 for norflurazon tolerance; a polynucleotide moleculeencoding acetohydroxyacid synthase (AHAS, aka ALS) described inSathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193 for tolerance tosulfonylurea herbicides; and the bar gene described in DeBlock, et al.(1987) EMBO J. 6:2513-2519 for glufosinate and bialaphos tolerance;resistant hydroxyphenyl pyruvate dehydrogenase (HPPD, U.S. Pat. No.6,768,044). The promoter of the present invention can express genes thatencode for phosphinothricin acetyltransferase, glyphosate resistantEPSPS, aminoglycoside phosphotransferase, hydroxyphenyl pyruvatedehydrogenase, hygromycin phosphotransferase, neomycinphosphotransferase, dalapon dehalogenase, bromoxynil resistantnitrilase, anthranilate synthase, glyphosate oxidoreductase andglyphosate-N-acetyl transferase.

Where plastid targeting is necessary, for example, the EPSPS enzymefunctions in a plant chloroplast, therefore, DNA molecules encoding achloroplast transit peptide (CTP) are engineered into a DNA moleculeencoding an EPSPS protein to encode a fusion protein of the CTP to the Nterminus of an EPSPS creating a chimeric molecule. A chimericpolynucleic acid coding sequence is comprised of two or more openreading frames joined in-frame that encode a chimeric protein, forexample, a chloroplast transit peptide and an EPSPS enzyme. A chimericgene refers to the multiple genetic elements derived from heterologoussources operably linked to comprise a gene. In the present invention theDNA construct expresses a chimeric CTP-EPSPS protein that directs theglyphosate resistant EPSPS protein into the plant chloroplast. In anative plant EPSPS gene, chloroplast transit peptide regions arecontained in the native coding sequence (for example, CTP2, Klee et al.,Mol. Gen. Genet. 210:47-442, 1987). The CTP is cleaved from the EPSPSenzyme at the chloroplast membrane to create a “mature EPSPS or EPSPSenzyme” that refers to the polypeptide sequence of the processed proteinproduct remaining after the chloroplast transit peptide has beenremoved. The production of glyphosate tolerant plants by expression of afusion protein comprising an amino-terminal CTP with a glyphosateresistant EPSPS enzyme is well known by those skilled in the art, (U.S.Pat. No. 5,627,061, U.S. Pat. No. 5,633,435, U.S. Pat. No. 5,312,910, EP0218571, EP 189707, EP 508909, and EP 924299). Those skilled in the artwill recognize that various chimeric constructs can be made that utilizethe functionality of a particular CTP to import glyphosate resistantEPSPS enzymes into the plant cell chloroplast.

In one embodiment of the invention, a polynucleotide molecule as shownin SEQ ID NO: 1-7 is incorporated into a construct such that apolynucleotide molecule of the present invention is operably linked to atranscribable polynucleotide molecule that is a gene of agronomicinterest. As used herein, the term “gene of agronomic interest” refersto a transcribable polynucleotide molecule that includes but is notlimited to a gene that provides a desirable characteristic associatedwith plant morphology, physiology, growth and development, yield,nutritional enhancement, disease or pest resistance, or environmental orchemical tolerance. The expression of a gene of agronomic interest isdesirable in order to confer an agronomically important trait. A gene ofagronomic interest that provides a beneficial agronomic trait to cropplants may be, for example, including, but not limited to geneticelements comprising herbicide resistance (U.S. Pat. No. 5,633,435 andU.S. Pat. No. 5,463,175), increased yield (U.S. Pat. No. 5,716,837),insect control (U.S. Pat. No. 6,063,597; U.S. Pat. No. 6,063,756; U.S.Pat. No. 6,093,695; U.S. Pat. No. 5,942,664; and U.S. Pat. No.6,110,464), fungal disease resistance (U.S. Pat. No. 5,516,671; U.S.Pat. No. 5,773,696; U.S. Pat. No. 6,121,436; U.S. Pat. No. 6,316,407,and U.S. Pat. No. 6,506,962), virus resistance (U.S. Pat. No. 5,304,730and U.S. Pat. No. 6,013,864), nematode resistance (U.S. Pat. No.6,228,992), bacterial disease resistance (U.S. Pat. No. 5,516,671),starch production (U.S. Pat. No. 5,750,876 and U.S. Pat. No. 6,476,295),modified oils production (U.S. Pat. No. 6,444,876), high oil production(U.S. Pat. No. 5,608,149 and U.S. Pat. No. 6,476,295), modified fattyacid content (U.S. Pat. No. 6,537,750), high protein production (U.S.Pat. No. 6,380,466), fruit ripening (U.S. Pat. No. 5,512,466), enhancedanimal and human nutrition (U.S. Pat. No. 5,985,605 and U.S. Pat. No.6,171,640), biopolymers (U.S. Pat. No. 5,958,745 and U.S. PatentPublication No. US20030028917), environmental stress resistance (U.S.Pat. No. 6,072,103), pharmaceutical peptides (U.S. Pat. No. 6,080,560),improved processing traits (U.S. Pat. No. 6,476,295), improveddigestibility (U.S. Pat. No. 6,531,648) low raffinose (U.S. Pat. No.6,166,292), industrial enzyme production (U.S. Pat. No. 5,543,576),improved flavor (U.S. Pat. No. 6,011,199), nitrogen fixation (U.S. Pat.No. 5,229,114), hybrid seed production (U.S. Pat. No. 5,689,041), andbiofuel production (U.S. Pat. No. 5,998,700). The genetic elements,methods, and transgenes described in the patents listed above are herebyincorporated by reference.

Alternatively, a transcribable polynucleotide molecule can effect theabove mentioned phenotypes by encoding a RNA molecule that causes thetargeted inhibition of expression of an endogenous gene, for example viaantisense, inhibitory RNA (RNAi), or cosuppression-mediated mechanisms.The RNA could also be a catalytic RNA molecule (i.e., a ribozyme)engineered to cleave a desired endogenous mRNA product. Thus, anypolynucleotide molecule that encodes a protein or mRNA that expresses aphenotype or morphology change of interest may be useful for thepractice of the present invention.

The constructs of the present invention are generally double Ti plasmidborder DNA constructs that have the right border (RB or AGRtu.RB) andleft border (LB or AGRtu.LB) regions of the Ti plasmid isolated fromAgrobacterium tumefaciens comprising a T-DNA, that along with transfermolecules provided by the Agrobacterium cells, permits the integrationof the T-DNA into the genome of a plant cell. The constructs alsocontain the plasmid backbone DNA segments that provide replicationfunction and antibiotic selection in bacterial cells, for example, anEscherichia coli origin of replication such as ori322, a broad hostrange origin of replication such as oriV or oriRi, and a coding regionfor a selectable marker such as Spec/Strp that encodes for Tn7aminoglycoside adenyltransferase (aadA) conferring resistance tospectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectablemarker gene. For plant transformation, the host bacterial strain isoften Agrobacterium tumefaciens ABI, C58, or LBA4404, however, otherstrains known to those skilled in the art of plant transformation canfunction in the present invention.

Transformed Plants and Plant Cells

As used herein, the term “transformed” refers to a cell, tissue, organ,or organism into which has been introduced a foreign polynucleotidemolecule, such as a construct. The introduced polynucleotide moleculemay be integrated into the genomic DNA of the recipient cell, tissue,organ, or organism such that the introduced polynucleotide molecule isinherited by subsequent progeny. A “transgenic” or “transformed” cell ororganism also includes progeny of the cell or organism and progenyproduced from a breeding program employing such a transgenic plant as aparent in a cross and exhibiting an altered phenotype resulting from thepresence of a foreign polynucleotide molecule. A plant transformationconstruct containing a promoter of the present invention may beintroduced into plants by any plant transformation method. Methods andmaterials for transforming plants by introducing a plant expressionconstruct into a plant genome in the practice of this invention caninclude any of the well-known and demonstrated methods includingelectroporation as illustrated in U.S. Pat. No. 5,384,253;microprojectile bombardment as illustrated in U.S. Pat. No. 5,015,580;U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.6,160,208; U.S. Pat. No. 6,399,861; and U.S. Pat. No. 6,403,865;Agrobacterium-mediated transformation as illustrated in U.S. Pat. No.5,824,877; U.S. Pat. No. 5,591,616; U.S. Pat. No. 5,981,840; and U.S.Pat. No. 6,384,301; and protoplast transformation as illustrated in U.S.Pat. No. 5,508,184, all of which are hereby incorporated by reference.

Methods for specifically transforming dicots are well known to thoseskilled in the art. Transformation and plant regeneration using thesemethods have been described for a number of crops including, but notlimited to, cotton (Gossypium hirsutum), soybean (Glycine max), peanut(Arachis hypogaea), and members of the genus Brassica.

Methods for transforming monocots are well known to those skilled in theart. Transformation and plant regeneration using these methods have beendescribed for a number of crops including, but not limited to, barley(Hordeum vulgarae); maize (Zea mays); oats (Avena sativa); orchard grass(Dactylis glomerata); rice (Oryza sativa, including indica and japonicavarieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp); tallfescue (Festuca arundinacea); turfgrass species (e.g. species: Agrostisstolonifera, Poa pratensis, Stenotaphrum secundatum); wheat (Triticumaestivum), and alfalfa (Medicago sativa). It is apparent to those ofskill in the art that a number of transformation methodologies can beused and modified for production of stable transgenic plants from anynumber of target crops of interest.

The transformed plants are analyzed for the presence of the genes ofinterest and the expression level and/or profile conferred by thepromoters of the present invention. Those of skill in the art are awareof the numerous methods available for the analysis of transformedplants. For example, methods for plant analysis include, but are notlimited to Southern blots or northern blots, PCR-based approaches,biochemical analyses, phenotypic screening methods, field evaluations,and immunodiagnostic assays.

The seeds of this invention can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plants ofthis invention including hybrid plant lines comprising the construct ofthis invention and expressing a gene of agronomic interest.

The present invention also provides for parts of the plants of thepresent invention. Plant parts, without limitation, include seed,endosperm, ovule and pollen. In a particularly preferred embodiment ofthe present invention, the plant part is a seed.

Still yet another aspect of the invention is a method of inhibiting weedgrowth in a field of transgenic crop plants comprising first plantingthe transgenic plants transformed with an expression cassette comprisingan isolated polynucleotide molecule having gene regulatory activity andcomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO: 1-7 and operably linked to a DNA molecule encoding aglyphosate tolerance gene and then applying glyphosate to the field atan application rate that inhibits the growth of weeds, wherein thegrowth and yield of the transgenic crop plant is not substantiallyaffected by the glyphosate application. The glyphosate application rateis the effective rate necessary to control weeds in a particularglyphosate tolerant crop; these rates may range from 8 ounces/acre to256 ounces/acre, preferably 16 ounces/acre to 128 ounces/acre, and morepreferably 32 ounces/acre to 96 ounces/acre. The glyphosate is appliedat least once during the growth of the glyphosate tolerant crop and maybe applied 2, 3, or 4 times during the growth of the crop or more asnecessary to control weeds in the field.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings is to be interpreted as illustrative and not in a limitingsense.

EXAMPLES Example 1 Constitutive Gene Identification

Four actin promoters were isolated from rice and two actin promoterswere cloned from maize. Genomic DNA was generated from corn (Zea mays)and rice (Oryza sativa) tissue using standard methods familiar to oneskilled in the art. The genomic libraries were prepared according tomanufacturer instructions (GenomeWalker™, CLONTECH Laboratories, Inc,Palo Alto, Calif.). In separate reactions, genomic DNA was subjected torestriction enzyme digestion overnight at 37° C. with the followingblunt-end endonucleases: EcoRV, Seal, DraI, PvuII, or StuI (CLONTECHLaboratories, Inc. Palo Alto, Calif.). The reaction mixtures wereextracted with phenol:chloroform, ethanol precipitated, and resuspendedin 10 mM Tris buffer pH 8.5. The purified blunt-ended genomic DNAfragments were then ligated to the GenomeWalker™ adaptors. Ligation ofthe resulting DNA fragments to adaptors was done according tomanufacturer protocol. The GenomeWalker™ sublibraries were aliquoted andstored at −20° C.

Four rice actin genes were aligned to design a gene-specific primercapable of annealing to multiple actin gene sequences. Three corn actingenes were aligned to design a gene-specific primer capable of annealingto multiple actin genes. Genomic DNA from corn and rice ligated to theGenomeWalker™ adaptor was subjected to PCR amplification in separatereactions with either the rice or corn gene-specific primer and a primerdesigned to anneal to the GenomeWalker™ adaptor. Standard protocolsprovided by the manufacturer were used. Those of skill in the art areaware of the variations in PCR conditions including choice ofpolymerase, cycling conditions, and concentrations of the reactioncomponents. PCR products were cloned into pUC19 and the DNA insert wassequenced for each clone. The presence of an actin gene sequence in theinsert sequence was used to identify the upstream promoter region.

Example 2 Constructs

The isolated promoters were cloned into an appropriate plant expressionvector for the subsequent characterization of the promoter in plants.The DNA fragments resulting from the nested PCR amplification describedabove were isolated and gel purified using methods familiar to thoseskilled in the art. The purified DNA was digested with one or morerestriction endonuclease(s) to permit ligation into a suitable cloningor expression vector. The promoter fragments were incorporated into aplant expression vector by positioning the promoter fragments in linkagewith a reporter gene by restriction enzyme digestion and ligation usingmethods well known in the art. The purified DNA of the present inventionwas ligated as a NotUNcoI fragment into a vector containing thenecessary plant expression elements, including the GUS or At-CP4-EPSPStransgene operably linked to the promoter. An aliquot of the ligationreaction was transformed into a suitable E. coli host such as DH10B andthe cells were plated on selection medium (100 μg/ml spectinomycin).Bacterial transformants were selected, grown in liquid culture, and theplasmid DNA was isolated using a commercially available kit such as theQiaprep Spin Microprep Kit (Qiagen Corp., Valencia, Calif.). Purifiedplasmid containing the predicted insert size was DNA sequenced in bothdirections using the dye terminator method and oligonucleotide primersdesigned to anneal to the region of the vector bordering the promoterinsertion site. Additional oligonucleotide primers for further DNAsequencing of the promoter were then prepared based on the sequenceproduced from the results of the first DNA sequencing reaction. This wasrepeated until a full-length sequence of the isolated promoter wasproduced.

Example 3 Promoter Characterization in Transient Systems

Corn protoplasts transformed with a vector containing a promoteroperably liked to the GUS transgene were analyzed for GUS expressionlevels, measured by GUS activity as MU (pMol/ng of Total Protein). GUSexpression levels of the actin promoters in protoplasts were compared toGUS expression levels in protoplasts transformed with a vector havingthe Os-actin 1 (ract 1) promoter driving GUS and a vector having theOs-actin 2 promoter driving GUS. Protoplasts transformed with a vectorcontaining only the luciferase transgene (LUC) were used as a negativecontrol for GUS activity. Data are provided in Table 1.

TABLE 1 GUS activity measurements in corn protoplasts Promoter operablylinked to GUS Seq ID Num GUS activity P-e35S 0.380 P-Os-actin 1 0.383P-Os-actin 2 0.341 P-Os-Act15a SEQ ID NO: 1 0.479 P-Os-Act15b SEQ ID NO:2 0.336 P-Os-Act16 SEQ ID NO: 3 0.343 P-Os-Act18 SEQ ID NO: 4 0.322P-Os-Act31 SEQ ID NO: 5 0.958 P-Zm-Act31 SEQ ID NO: 6 1.156 P-Zm-Act33SEQ ID NO: 7 0.710 LUC 0.218

Corn protoplasts transformed with a vector containing a promoteroperably linked to the At-CP4-EPSPS transgene were analyzed for CP4expression levels, measured as CP4-EPSPS accumulation (ng of CP4/mg oftotal protein). CP4-EPSPS expression levels of the actin promoters inprotoplasts were compared to CP4-EPSPS expression levels in protoplaststransformed with a vector having the Os-actin 1 (ract1) promoter drivingAt-CP4-EPSPS and a vector having the e35S promoter driving At-CP4-EPSPS.Protoplasts transformed with a vector containing only the luciferasetransgene (LUC) were used as a negative control for GUS activity. Dataare provided in Table 2.

TABLE 2 CP4-EPSPS accumulation measurements in corn protoplasts Promoteroperably linked to At-CP4-EPSPS Related FIG. CP4-EPSPS accumulationP-e35S None 7.516 P-Os-actin 1 None 2.256 P-Os-actin 2 None 2.749P-Os-Act15a FIG. 1 0.850 P-Os-Act15b FIG. 2 2.505 P-Os-Act16 FIG. 31.227 P-Os-Act18 FIG. 4 1.792 P-Os-Act31 FIG. 5 4.588 P-Zm-Act31 FIG. 64.714 P-Zm-Act33 FIG. 7 0.504 LUC None 0.793

Example 4 Promoter Characterization in Transgenic plants

Transgenic corn plants transformed with a vector containing a promoteroperably linked to the CP4-EPSPS transgene were analyzed fortransformation efficiency, copy number, and transgene expression level.Transformants were analyzed for transformation efficiency measured asthe percent of explants produced (embryos that regenerated to formplants) compared to the total transformed. Genomic DNA from transgenicplants was used to determine the percent of single copy events out ofthe total number of transgenic plants analyzed. Single copy events wereused to determine CP4-EPSPS transgene expression levels, measured as thepercent of CP4-EPSPS accumulation relative to Roundup Ready® corn lineNK603. CP4-EPSPS expression levels of the actin promoters were comparedto CP4-EPSPS expression levels in transgenic plants transformed with avector having the Os-actin 1 (ract1) promoter driving CP4-EPSPS and avector having the e35S promoter driving CP4-EPSPS. Data are provided inTable 3.

TABLE 3 Measurements of transgenic corn plants Promoter operably linkedto Related Transformation Single Copy CP4-EPSPS CP4-EPSPS FIG.Efficiency Events accumulation P-e35S None 5.4% 70% 75.5% P-Os-Act1 None8.2% 31% 78.1% P-Os-Act2 None   0% N/A N/A P-Os-Act15a FIG. 1 5.3% 59%71.7% P-Os-Act16 FIG. 3 5.4% 52% 76.9% P-Os-Act18 FIG. 4 5.2% 32% 48.7%P-Os-Act31 FIG. 5 6.6% 38% 50.5% P-Zm-Act31 FIG. 6 4.5% 57% 66.2%P-Zm-Act33 FIG. 7   0% N/A N/A P-Os-Act1/Pe35S None N/A N/A  100%

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims. All publications and publishedpatent documents cited in this specification are incorporated herein byreference to the same extent as if each individual publication or patentapplication is specifically and individually indicated to beincorporated by reference.

1. A polynucleotide molecule having promoter activity and comprising apolynucleotide sequence comprising SEQ ID NO: 3, wherein saidpolynucleotide is operably linked to a transcribable heterologouspolynucleotide sequence.
 2. The polynucleotide molecule of claim 1,wherein said transcribable polynucleotide molecule is a marker gene. 3.The polynucleotide molecule of claim 1, wherein said transcribablepolynucleotide molecule is a gene of agronomic interest.
 4. Thepolynucleotide molecule of claim 3, wherein said gene of agronomicinterest is a herbicide tolerance gene selected from the groupconsisting of genes that encode for phosphinothricin acetyltransferase,glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase,hydroxyphenyl pyruvate dehydrogenase, dalapon dehalogenase, bromoxynilresistant nitrilase, anthranilate synthase, glyphosate oxidoreductaseand glyphosate-N-acetyl transferase.
 5. A transgenic plant stablytransformed with the polynucleotide molecule of claim
 1. 6. Thetransgenic plant of claim 5, wherein said plant is a monocotyledonousplant selected from the group consisting of wheat, maize, rye, rice,oat, barley, turfgrass, sorghum, millet and sugarcane.
 7. The transgenicplant of claim 5, wherein said plant is a dicotyledonous plant selectedfrom the group consisting of tobacco, tomato, potato, soybean, cotton,canola, sunflower and alfalfa.
 8. A seed of said transgenic plant ofclaim 6, wherein the seed comprises said polynucleotide molecule.
 9. Aseed of said transgenic plant of claim 7, wherein the seed comprisessaid polynucleotide molecule.
 10. A method of inhibiting weed growth ina field of transgenic glyphosate tolerant crop plants comprising: i)planting the transgenic plants transformed with an expression cassettecomprising a polynucleotide molecule of claim 1 operably linked to a DNAmolecule encoding a glyphosate tolerance gene and ii) applyingglyphosate to the field at an application rate that inhibits the growthof weeds, wherein the growth and yield of the transgenic crop plant isnot substantially affected by the glyphosate application.
 11. The methodof claim 10, wherein said glyphosate tolerance gene is selected from thegroup consisting of a gene encoding for a glyphosate resistant5-enolpyruvylshikimate-3-phosphate synthase, a glyphosateoxidoreductase, and a glyphosate-N-acetyltransferase.
 12. The method ofclaim 10, wherein the transgenic plants are capable of tolerating anapplication rate up to 256 ounces/acre.
 13. The method of claim 10,wherein the transgenic plants are capable of tolerating an applicationrate ranging from 8 ounces/acre to 128 ounces/acre.
 14. The method ofclaim 10, wherein the transgenic plants are capable of tolerating anapplication rate ranging from 32 ounces/acre to 96 ounces/acre.
 15. Themethod of claim 10, wherein the application of glyphosate is at leastonce during the growth of the crop.